RESUMO
SHQ1 was reported to control the biogenesis and assembly of H/ACA ribonucleoprotein particles (RNPs). It was independently isolated as a growth suppressor, GRIM1, in a genetic screen. Recent studies have indicated that SHQ1 inhibits prostate cancer growth and metastasis. SHQ1 facilitates MYC RNA splicing to promote T-acute lymphoblastic leukemia (T-ALL) development. Thus, the mechanisms of SHQ1 in cancers remain largely unknown. We report here that SHQ1 promotes tumor apoptosis and chemo-sensitivity in hepatocellular carcinoma (HCC) cells. In HCC tissues from patients, expression of SHQ1 was significantly decreased in the tumor compared to adjacent tissues. Experiments with HCC xenograft models revealed that restoring SHQ1 levels enhanced the anti-tumor activity of the endoplasmic reticulum (ER) stress inducer tunicamycin (TM) and common chemotherapy drug paclitaxel (PTX). Mechanistically, SHQ1 is an ER-stress response gene which is regulated by p50ATF6 and XBP1s through an ER stress response like element located on the SHQ1 promoter. SHQ1 interacts with the ER chaperone GRP78 to release ER sensors PERK/IRE1α/ATF6 from GRP78/ER-sensor complexes, leading to hyper-activation of unfolded protein response (UPR). In the persistent ER stress conditions of a HepG2 xenograft tumor model, SHQ1-mediated hyper-activation of ER-sensor signaling induces apoptosis. Our study thus demonstrates a SHQ1-mediated ER-stress response feedback loop that promotes tumor sensitivity to chemotherapeutics.
Assuntos
Antineoplásicos/farmacologia , Apoptose , Estresse do Retículo Endoplasmático/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Ligação Competitiva/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Feminino , Proteínas de Choque Térmico/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Biológicos , Paclitaxel/farmacologia , Ligação Proteica/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Resposta a Proteínas não Dobradas/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Melanoma is one of the most lethal skin malignancies in the world. Interferons (IFNs) have been also demonstrated in response to tumor cell and IFNs such as IFN-α have been used for melanoma treatment. The long chain double-stranded RNA (dsRNA) (from a variety of nonviral sources) is a potent activator of the IFN system and an inducer of cell apoptosis. Panaxadiolsaponins (PDS) is a major Panax ginseng-derived active component with known antitumor activity and immune modulation. Here, we investigated a hypothesis that the combination of PDS and total natural dsRNA (as opposed to the synthetic dsRNA) will suppress tumor growth better than the individual agents. We have evaluated the antitumor and immunostimulatory effects of the combination of natural long chain dsRNA (derived from yeast) and PDS on melanoma cell line B16 and mice xenograft model. The underlying mechanisms of growth suppression were investigated by analyzing dsRNA-activated pathways, apoptosis, and cell cycle. Natural dsRNA and PDS exert superior anticancer effects than either agent alone. Natural dsRNA and PDS combination might be a promising strategy for treating malignancies, including melanoma.
Assuntos
Antineoplásicos/farmacologia , Produtos Biológicos/farmacologia , Melanoma Experimental/tratamento farmacológico , RNA de Cadeia Dupla/farmacologia , Sapogeninas/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Melanoma Experimental/patologia , Camundongos , Panax/química , Células Tumorais CultivadasRESUMO
Studies suggest that tunicamycin may work as a therapeutic drug to cancer cells by inducing stress in the endoplasmic reticulum (ER) through unfolded protein response (UPR) and thereby promoting apoptosis. However, mechanisms of the prolonged activation of the UPR under sustained ER stress in the regulation of cell apoptosis are largely unknown. To delineate the role of candidate genes in the apoptotic process under ER stress and to search for new therapeutic strategies to treat metastatic castration resistant prostate cancer, we performed whole genome expression microarray analysis in tunicamycin treated metastatic androgen-insensitive prostate cancer cells, PC-3. Among several induced genes, the expression of eNOS (NOS3) gene was remarkably high. The increased expression of eNOS activates mTORC1 through RagC. This results into an accumulation of p62 (SQSTM1) which facilitates aggregation of ubiquitinated protein thus compromising clearance of misfolded toxic protein aggregates. Lastly, association of p62 proteins and misfolded proteins promote reactive oxygen species (ROS) mediated mitochondrial apoptosis. Overall, our data demonstrate that tunicamycin induced ER stress promotes prostate cancer cell death by activating mTORC1 through eNOS-RagC pathway.
RESUMO
p16INK4A and p53 are two important tumor suppressor proteins that play essential roles during cell proliferation and aging through regulating the expression of several genes. Here, we report that p16INK4A and p53 co-regulate a plethora of transcripts. Furthermore, both proteins colocalize in the nucleus of human primary skin fibroblasts and breast luminal cells, and form a heteromer whose level increases in response to genotoxic stress as well as aging of human fibroblasts and various mouse organs. CDK4 is also present in this heteromeric complex, which is formed only in the presence of DNA both in vitro using pure recombinant proteins and in vivo. We have also shown that p16INK4A enhances the binding efficiency of p53 to its cognate sequence presents in the CDKN1A promoter in vitro, and both proteins are present at the promoters of CDKN1A and BAX in vivo. Importantly, the fourth ankyrin repeat of p16INK4A and the C-terminal domain of p53 were necessary for the physical association between these two proteins. The physiologic importance of this association was revealed by the inability of cancer-associated p16INK4A mutants to interact with p53 and to transactivate the expression of its major targets CDKN1A and BAX in the p16-defective U2OS cells expressing either wild-type or mutated p16INK4A . Furthermore, the association between p16INK4A and p53 was capital for their nuclear colocalization, the X-ray-dependent induction of p21 and Bax proteins as well as the induction of apoptosis in various types of cells. Together, these results show DNA-dependent physical interaction between p16INK4A and p53.
Assuntos
Apoptose , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , DNA/metabolismo , Regulação da Expressão Gênica , Proteína Supressora de Tumor p53/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , DNA/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Transcrição Gênica , Proteína Supressora de Tumor p53/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Expression of DAPK1, a critical regulator of autophagy and apoptosis, is lost in a wide variety of tumors, although the mechanisms are unclear. A transcription factor complex consisting of ATF6 (an endoplasmic reticulum-resident factor) and C/EBP-ß is required for the IFN-γ-induced expression of DAPK1 IFN-γ-induced proteolytic processing of ATF6 and phosphorylation of C/EBP-ß are obligatory for the formation of this transcriptional complex. We report that defects in this pathway fail to control growth of chronic lymphocytic leukemia (CLL). Consistent with these observations, IFN-γ and chemotherapeutics failed to activate autophagy in CLL patient samples lacking ATF6 and/or C/EBP-ß. Together, these results identify a molecular basis for the loss of DAPK1 expression in CLL.
Assuntos
Fator 6 Ativador da Transcrição/metabolismo , Autofagia , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteínas Quinases Associadas com Morte Celular/biossíntese , Regulação Enzimológica da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/metabolismo , Proteínas de Neoplasias/metabolismo , Fator 6 Ativador da Transcrição/genética , Proteína beta Intensificadora de Ligação a CCAAT/genética , Linhagem Celular Transformada , Proteínas Quinases Associadas com Morte Celular/genética , Feminino , Humanos , Interferon gama/farmacologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Masculino , Proteínas de Neoplasias/genéticaRESUMO
Nicotine, a main addictive compound in tobacco smoke, has been linked to promotion and progression of lung, head and neck, pancreatic, and breast cancers, but the detailed mechanisms of cancer progression remain elusive. Here, we show that nicotine induces the expression of galectin-3 (an anti-apoptotic ß-galactoside-binding lectin) in breast cancer cell line and in primary tumors from breast cancer patients. Nicotine-induced up regulation of galectin-3 is due to an increased expression of α9 isoform of nicotinic acetylcholine receptor (α9nAChR), which activates transcription factor STAT3 that in turn, physically binds to galectin-3 (LGALS3) promoter and induces transcription of galectin-3. Intracellular galectin-3 increased mitochondrial integrity and suppressed chemotherapeutic-induced apoptosis of breast cancer cell. Moreover, nicotine-induced enrichment of side population cells with cancer stem cell-like properties was modulated by galectin-3 expression and could be significantly reduced by transient knock down of LGALS3 and its upstream signaling molecules STAT3 and α9nAChR. Thus, galectin-3 or its upstream signaling molecule STAT3 or α9nAChR could be a potential target to prevent nicotine-induced chemoresistance in breast cancer.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Células-Tronco Neoplásicas/metabolismo , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Fragmentação do DNA , Galectina 3/biossíntese , Humanos , Imuno-Histoquímica , Células-Tronco Neoplásicas/patologia , Reação em Cadeia da Polimerase em Tempo Real , Receptores Nicotínicos/metabolismo , Fator de Transcrição STAT3/metabolismo , Células da Side Population , TransfecçãoRESUMO
p16(INK4a) is a tumor suppressor protein involved in several stress-related cellular responses, including apoptosis. Recent lines of evidence indicate that p16(INK4a) is also a modulator of gene expression. However, the molecular mechanisms underlying this novel function are still obscure. Here, we present clear evidence that p16(INK4a) modulates the levels of various microRNAs, with marked positive effect on miR-141 and miR-146b-5p. This effect is mediated through the formation of the p16-CDK4-Sp1 heterocomplex, which binds to Sp1 consensus-binding motifs present in the promoters of miR-141 and miR-146b-5p, and it enables their transcription. In addition, we have shown that p16(INK4a) interacts with Sp1 through the fourth ankyrin repeat, which is crucial for Sp1 binding to the miR-141 and miR-146b-5p promoters and their transcriptional activation. The physiological importance of this association was revealed by the inability of cancer-related p16(INK4a) mutants to interact with Sp1. Moreover, we have shown p16-CDK4-Sp1-dependent up-regulation of miR-141 and miR-146b-5p following UV light-induced DNA damage and the role of these two microRNAs in mediating p16-related induction of apoptosis in response to this genotoxic stress. Together, these results indicate that p16(INK4a) associates with CDK4 not only to inhibit the cell cycle but also to enable the transcription of two important onco-microRNAs, which act as downstream effectors.
Assuntos
Quinase 4 Dependente de Ciclina/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Fator de Transcrição Sp1/metabolismo , Sequência de Bases , Ciclo Celular , Linhagem Celular , Quinase 4 Dependente de Ciclina/química , Inibidor p16 de Quinase Dependente de Ciclina/química , Dano ao DNA , Humanos , Dados de Sequência Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Estabilidade de RNA , Fator de Transcrição Sp1/química , Ativação Transcricional/efeitos da radiação , Raios Ultravioleta/efeitos adversosRESUMO
Gene-associated with retinoid-interferon induced mortality-19 (GRIM-19), a STAT3-inhibitory protein, was isolated as a growth-suppressive gene product using a genome-wide expression knockdown screen. We and others have shown a loss of expression and occurrence of mutations in the GRIM-19 gene in a variety of primary human cancers, indicating its potential role as tumor suppressor. To help investigate its role in tumor development in vivo, we generated a genetically modified mouse in which Grim-19 can be conditionally inactivated. Deletion of Grim-19 in the skin significantly increased the susceptibility of mice to chemical carcinogenesis, resulting in development of squamous cell carcinomas. These tumors had high Stat3 activity and an increased expression of Stat3-responsive genes. Loss of Grim-19 also caused mitochondrial electron transport dysfunction resulting from failure to assemble electron transport chain complexes and altered the expression of several cellular genes involved in glycolysis. Surprisingly, the deletion of a single copy of the Grim-19 gene was sufficient to promote carcinogenesis and formation of invasive squamous cell carcinomas. These observations highlight the critical role of GRIM-19 as a tumor suppressor.
Assuntos
Carcinogênese/genética , Carcinoma de Células Escamosas/genética , NADH NADPH Oxirredutases/genética , Animais , Primers do DNA/genética , Componentes do Gene , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Vetores Genéticos/genética , Imuno-Histoquímica , Camundongos , Camundongos Knockout , NADH NADPH Oxirredutases/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Fator de Transcrição STAT3/metabolismo , Análise de Sequência de RNARESUMO
Lung epithelial and endothelial cell death caused by pro-oxidant insults is a cardinal feature of acute lung injury/acute respiratory distress syndrome (ALI/ARDS) patients. The NF-E2-related factor 2 (NRF2) activation in response to oxidant exposure is crucial to the induction of several antioxidative and cytoprotective enzymes that mitigate cellular stress. Since prolonged exposure to hyperoxia causes cell death, we hypothesized that chronic hyperoxia impairs NRF2 activation, resulting in cell death. To test this hypothesis, we exposed nonmalignant small airway epithelial cells (AECs) to acute (1-12 h) and chronic (36-48 h) hyperoxia and evaluated cell death, NRF2 nuclear accumulation and target gene expression, and NRF2 recruitment to the endogenous HMOX1 and NQO1 promoters. As expected, hyperoxia gradually induced death in AECs, noticeably and significantly by 36 h; ~60% of cells were dead by 48 h. However, we unexpectedly found increased expression levels of NRF2-regulated antioxidative genes and nuclear NRF2 in AECs exposed to chronic hyperoxia as compared to acute hyperoxia. Chromatin Immunoprecipitation (ChIP) assays revealed an increased recruitment of NRF2 to the endogenous HMOX1 and NQO1 promoters in AECs exposed to acute or chronic hyperoxia. Thus, our findings demonstrate that NRF2 activation and antioxidant gene expression are functional during hyperoxia-induced lung epithelial cell death and that chronic hyperoxia does not impair NRF2 signaling overall.
Assuntos
Antioxidantes/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Hiperóxia/metabolismo , Hiperóxia/patologia , Pulmão/patologia , Fator 2 Relacionado a NF-E2/metabolismo , Morte Celular/genética , Núcleo Celular/metabolismo , Sobrevivência Celular/genética , Regulação da Expressão Gênica , Heme Oxigenase-1/genética , Humanos , NAD(P)H Desidrogenase (Quinona)/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica/genéticaRESUMO
The interferon (IFN) family of cytokines regulates many cellular processes, such as transcription, translation, post-translational modifications, and protein degradation. IFNs induce growth inhibition and/or cell death, depending on the cell type, by employing different proteins. This review describes a novel growth-suppressive pathway employed by IFNs that affects rRNA levels. Maturation of rRNA involves numerous noncoding small regulatory RNA-guided processes. These regulatory RNAs, called small nucleolar RNA (snoRNAs), function as a ribonucleoprotein particle (RNP) in the nucleolus. The biogenesis of snoRNPs is dependent on core protein and assembly factors. Our laboratory recently isolated a growth-suppressive protein gene associated with retinoid-IFN-induced mortality (GRIM)-1 using a genetic screen. IFN-inducible GRIM-1 (SHQ1) is an assembly factor that controls one arm of the snoRNP machinery. GRIM-1 inhibits sno/scaRNP formation to induce growth suppression via reduction in mature rRNA levels. Loss of GRIM-1 observed in certain cancers implicates it to be a novel tumor suppressor. Certain snoRNAs have been reported to act as either oncogenes or tumor suppressors in vitro. Recent studies have shown that certain sno/scaRNAs are further processed into micro RNA-like molecules to control translation of protein-coding RNAs. We present a model as to how these small regulatory RNAs influence cell growth and a potential role for GRIM-1 in this process.
Assuntos
Interferons/metabolismo , Neoplasias/metabolismo , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Transporte/metabolismo , Humanos , Interferons/genética , Peptídeos e Proteínas de Sinalização Intracelular , Neoplasias/genética , Região Organizadora do Nucléolo/genética , Região Organizadora do Nucléolo/metabolismo , RNA Ribossômico/biossínteseRESUMO
Cancer metastasis and immune suppression are critical issues in cancer therapy. Here, we show that a ß-galactoside-binding lectin [galectin-3 (gal3)] that recognizes the Thomsen-Friedenreich disaccharide (TFD, Galß1,3GalNAc) present on the surface of most cancer cells is involved in promoting angiogenesis, tumor-endothelial cell adhesion, and metastasis of prostate cancer cells, as well as evading immune surveillance through killing of activated T cells. To block gal3-mediated interactions, we purified a glycopeptide from cod (designated TFD100) that binds gal3 with picomolar affinity. TFD100 blocks gal3-mediated angiogenesis, tumor-endothelial cell interactions, and metastasis of prostate cancer cells in mice at nanomolar levels. Moreover, apoptosis of activated T cells induced by either recombinant gal3 or prostate cancer patient serum-associated gal3 was inhibited at nanomolar concentration of TFD100. Because the gal3-TFD interaction is a key factor driving metastasis in most epithelial cancers, this high-affinity TFD100 should be a promising antimetastatic agent for the treatment of various cancers, including prostate adenocarcinoma.
Assuntos
Adenocarcinoma/tratamento farmacológico , Proteínas Anticongelantes/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Peixes/farmacologia , Gadus morhua , Galectina 3/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Linfócitos T/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Proteínas Anticongelantes/química , Proteínas Anticongelantes/isolamento & purificação , Antígenos Glicosídicos Associados a Tumores/química , Antígenos Glicosídicos Associados a Tumores/metabolismo , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Proteínas de Peixes/química , Proteínas de Peixes/isolamento & purificação , Células Endoteliais da Veia Umbilical Humana , Humanos , Células Jurkat , Masculino , Camundongos , Metástase Neoplásica , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologiaRESUMO
SerpinB2 or plasminogen activator inhibitor type 2 (PAI-2) is highly induced in macrophages in response to inflammatory stimuli and is linked to the modulation of innate immunity, macrophage survival, and inhibition of plasminogen activators. Lipopolysaccharide (LPS), a potent bacterial endotoxin, can induce SerpinB2 expression via the toll-like receptor 4 (TLR4) by â¼1000-fold over a period of 24 hrs in murine macrophages. To map the LPS-regulated SerpinB2 promoter regions, we transfected reporter constructs driven by the â¼5 kb 5'-flanking region of the murine SerpinB2 gene and several deletion mutants into murine macrophages. In addition, we compared the DNA sequence of the murine 5' flanking sequence with the sequence of the human gene for homologous functional regulatory elements and identified several regulatory cis-acting elements in the human SERPINB2 promoter conserved in the mouse. Mutation analyses revealed that a CCAAT enhancer binding (C/EBP) element, a cyclic AMP response element (CRE) and two activator protein 1 (AP-1) response elements in the murine SerpinB2 proximal promoter are essential for optimal LPS-inducibility. Electrophoretic mobility shift (EMSA) and chromatin immunoprecipitation (ChIP) assays demonstrated that LPS induces the formation of C/EBP-ß containing complexes with the SerpinB2 promoter. Importantly, both constitutive and LPS-induced SerpinB2 expression was severely abrogated in C/EBP-ß-null mouse embryonic fibroblasts (MEFs) and primary C/EBP-ß-deficient peritoneal macrophages. Together, these data provide new insight into C/EBP-ß-dependent regulation of inflammation-associated SerpinB2 expression.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Regulação da Expressão Gênica , Lipopolissacarídeos/metabolismo , Inibidor 2 de Ativador de Plasminogênio/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Sequência Conservada , Análise Mutacional de DNA , Fibroblastos/citologia , Humanos , Macrófagos/metabolismo , Camundongos , Dados de Sequência Molecular , Regiões Promotoras GenéticasRESUMO
OBJECTIVES: To investigate the therapeutic utility of an attenuated bacterium carrying a plasmid that co-expresses Endostatin, an inhibitor of tumor neovasculogenesis, and a shRNA that targets Stat3 to suppress prostate cancer growth. METHODS: Plasmid pEndo-Si-Stat3 was constructed and introduced into an attenuated strain of Salmonella enterica serovar typhimurium. The resultant recombinant bacterium was used as a vector to deliver the plasmid to tumor cells growing in vivo. Tumor-associated gene and protein expression changes were measured by using RT-PCR and Western blot analyses. Expression of Endostatin in tumor tissue was detected by ELISA. The presence of vector bacteria in tissues was monitored and tumor destruction was assessed by using TUNEL and H&E staining assays. RESULTS: Bacterially delivered pEndo-Si-Stat3 decreased Stat3 levels and increased Endostatin expression in mouse tumors, resulting in a significant suppression of tumor growth (P < 0.01). Expression of Bcl-2 and PCNA was down-regulated and Caspase3 expression was up-regulated to promote apoptosis of tumor cells. CONCLUSIONS: Successful delivery by attenuated Salmonella of the combination therapeutic plasmid simultaneously knocked down the expression of Stat3 and resulted in over-expression of Endostatin, which synergistically inhibited prostate cancer growth.
Assuntos
Endostatinas/genética , Técnicas de Transferência de Genes , Neoplasias da Próstata/terapia , RNA Interferente Pequeno/genética , Fator de Transcrição STAT3/genética , Animais , Antígenos CD34/metabolismo , Apoptose/genética , Western Blotting , Linhagem Celular Tumoral , Terapia Combinada , Endostatinas/metabolismo , Expressão Gênica , Terapia Genética , Vetores Genéticos/genética , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasmídeos/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/metabolismo , Salmonella typhimurium/genética , Fatores de Tempo , Carga Tumoral/genética , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The signal transducer and activator of transcription 3 (STAT3) protein is critical for multiple cytokine and growth factor-induced biological responses in vivo. Its transcriptional activity is controlled by a transient phosphorylation of a critical tyrosine. Constitutive activation of STAT3 imparts resistance to apoptosis, promotes cell proliferation, and induces de novo micro-angiogenesis, three of the six cardinal hallmarks of a typical cancer cell. Earlier we reported the isolation of GRIM-19 as a growth suppressor using a genome-wide expression knockdown strategy. GRIM-19 binds to STAT3 and suppresses its transcriptional activity. To understand the pathological relevance of GRIM-19, we screened a set of primary head and neck tumors and identified three somatic mutations in GRIM-19. Wild-type GRIM-19 suppressed cellular transformation by a constitutively active form of STAT3, whereas tumor-derived mutants L71P, L91P and A95T significantly lost their ability to associate with STAT3, block gene expression, and suppress cellular transformation and tumor growth in vivo. Additionally, these mutants lost their capacity to prevent metastasis. These mutations define a mechanism by which STAT3 activity is deregulated in certain human head and neck tumors.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Regulação Neoplásica da Expressão Gênica , Chaperonas Moleculares/metabolismo , Mutação , NADH NADPH Oxirredutases/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Nus , Neoplasias Bucais/metabolismo , Metástase Neoplásica , Ratos , Transcrição GênicaRESUMO
The E6 protein of the oncogenic HPV-16 functions by interfering with the normal cell cycle control mechanisms, particularly those controlled by p53. In this study, we developed a dual expression plasmid that coexpressed-E6-specific siRNA and wild type p53, and to evaluate its effects on cervical cancer growth. We found that simultaneous expression of pSi-E6-P53 caused a robust suppression of tumor growth when compared to the controls either E6-specific siRNA or p53 alone. In conclusion, our findings demonstrate that a combined strategy of co-expressed E6-specific siRNA and p53 synergistically and more effectively suppressed cervical tumor growth when compared with single treatment.
Assuntos
Proliferação de Células , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/terapia , RNA Interferente Pequeno/genética , Proteínas Repressoras/genética , Proteína Supressora de Tumor p53/metabolismo , Neoplasias do Colo do Útero/terapia , Animais , Apoptose , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Sobrevivência Celular , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Terapia Genética/métodos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/patologia , Plasmídeos/genética , Proteínas Repressoras/metabolismo , Salmonella typhimurium/genética , Transformação Bacteriana , Carga Tumoral , Proteína Supressora de Tumor p53/genética , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologiaRESUMO
The IFN family of cytokines operates a frontline defense against pathogens and neoplastic cells in vivo by controlling the expression of several genes. The death-associated protein kinase 1 (DAPK1), an IFN-γ-induced enzyme, controls cell cycle, apoptosis, autophagy, and tumor metastasis, and its expression is frequently down-regulated in a number of human tumors. Although the biochemical action of DAPK1 is well understood, mechanisms that regulate its expression are unclear. Previously, we have shown that transcription factor C/EBP-ß is required for the basal and IFN-γ-induced expression of DAPK1. Here, we show that ATF6, an ER stress-induced transcription factor, interacts with C/EBP-ß in an IFN-stimulated manner and is obligatory for Dapk1 expression. IFN-stimulated proteolytic processing of ATF6 and ERK1/2-mediated phosphorylation of C/EBP-ß are necessary for these interactions. More importantly, IFN-γ failed to activate autophagic response in cells lacking either ATF6 or C/EBP-ß. Consistent with these observations, the Atf6(-/-) mice were highly susceptible to lethal bacterial infections compared with the wild-type mice. These studies not only unravel an IFN signaling pathway that controls cell growth and antibacterial defense, but also expand the role of ATF6 beyond ER stress.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/fisiologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Interferon gama/fisiologia , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteínas Quinases Associadas com Morte Celular , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Proteólise , Fatores de Transcrição/metabolismoRESUMO
PURPOSE: Death-associated protein kinase 1 (DAPK1), a tumor suppressor, is a rate-limiting effector in an endoplasmic reticulum (ER) stress-dependent apoptotic pathway. Its expression is epigenetically suppressed in several tumors. A mechanistic basis for epigenetic/transcriptional repression of DAPK1 was investigated in certain forms of acute myeloid leukemia (AML) with poor prognosis, which lacked ER stress-induced apoptosis. EXPERIMENTAL DESIGN: Heterogeneous primary AMLs were screened to identify a subgroup with Flt3ITD in which repression of DAPK1, among NF-κB-and c-Jun-responsive genes, was studied. RNA interference knockdown studies were carried out in an Flt3ITD(+) cell line, MV-4-11, to establish genetic epistasis in the pathway Flt3ITD-TAK1-DAPK1 repression, and chromatin immunoprecipitations were carried out to identify proximate effector proteins, including TAK1-activated p52NF-κB, at the DAPK1 locus. RESULTS: AMLs characterized by normal karyotype with Flt3ITD were found to have 10- to 100-fold lower DAPK1 transcripts normalized to the expression of c-Jun, a transcriptional activator of DAPK1, as compared with a heterogeneous cytogenetic category. In addition, Meis1, a c-Jun-responsive adverse AML prognostic gene signature was measured as control. These Flt3ITD(+) AMLs overexpress relB, a transcriptional repressor, which forms active heterodimers with p52NF-κB. Chromatin immunoprecipitation assays identified p52NF-κB binding to the DAPK1 promoter together with histone deacetylase 2 (HDAC2) and HDAC6 in the Flt3ITD(+) human AML cell line MV-4-11. Knockdown of p52NF-κB or its upstream regulator, NF-κB-inducing kinase (NIK), de-repressed DAPK1. DAPK1-repressed primary Flt3ITD(+) AMLs had selective nuclear activation of p52NF-κB. CONCLUSIONS: Flt3ITD promotes a noncanonical pathway via TAK1 and p52NF-κB to suppress DAPK1 in association with HDACs, which explains DAPK1 repression in Flt3ITD(+) AML.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Leucemia Mieloide Aguda/metabolismo , Subunidade p52 de NF-kappa B/metabolismo , Transdução de Sinais , Tirosina Quinase 3 Semelhante a fms/metabolismo , Apoptose , Proteínas Reguladoras de Apoptose/genética , Sítios de Ligação , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Metilação de DNA , Proteínas Quinases Associadas com Morte Celular , Epigênese Genética , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Células HL-60 , Humanos , Cariótipo , Leucemia Mieloide Aguda/genética , MAP Quinase Quinase Quinases/metabolismo , Mutação , Regiões Promotoras Genéticas , Ligação Proteica , Sequências de Repetição em Tandem , Células Tumorais Cultivadas , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
We have recently isolated novel IFN-inducible gene, Gene associated with Retinoid-Interferon-induced Mortality-1 (GRIM-1), using a genetic technique. Moderate ectopic expression of GRIM-1 caused growth inhibition and sensitized cells to retinoic acid (RA)/IFN-induced cell death while high expression caused apoptosis. GRIM-1 depletion, using RNAi, conferred a growth advantage. Three protein isoforms (1α, 1ß and 1γ) with identical C-termini are produced from GRIM-1 mRNA. We show that GRIM-1 isoforms interact with NAF1 and DKC1, two essential proteins required for box H/ACA sno/sca RNP biogenesis and suppresses box H/ACA RNA levels in mammalian cells by delocalizing NAF1. Suppression of these small RNAs manifests as inefficient rRNA maturation and growth suppression. Interestingly, yeast Shq1p also caused growth suppression in mammalian cells. Consistent with its growth-suppressive property, GRIM-1 expression is lost in a number of human primary prostate tumors. Our observations support a recent study that GRIM-1 might act as a co-tumor suppressor in the prostate.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Regulação Neoplásica da Expressão Gênica , RNA Ribossômico/genética , RNA Nucleolar Pequeno/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/genética , Linhagem Celular Tumoral , Células HeLa , Humanos , Imuno-Histoquímica , Interferon beta/farmacologia , Masculino , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Ribossômico/metabolismo , RNA Nucleolar Pequeno/metabolismo , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Tretinoína/farmacologiaRESUMO
BACKGROUND: Our previous studies showed a down-regulation of GRIM-19 in primary human cervical cancers, and restoration of GRIM-19 induced tumor regression. The induction of tumor suppressor protein p53 ubiquitination and degradation by E6 oncoportein of high risk-HPV through forming a stable complex with E6AP is considered as a critical mechanism for cervical tumor development. The aims of this study were to determine the potential role of GRIM-19 in rescuing p53 protein and inducing cervical cancer cell apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: The protein levels of GRIM-19 and p53 were detected in normal cervical tissues from 45 patients who underwent hysterectomy for reasons other than neoplasias of either the cervix or endometrium, and cervical cancer tissues from 60 patients with non-metastatic squamous epithelial carcinomas. Coimmunoprecipitation and GST pull-down assay were performed to examine the interaction of GRIM-19 with 18E6 and E6AP in vivo and in vitro respectively. The competition of 18E6 with E6AP in binding GRIM-19 by performing competition pull-down assays was designed to examine the disruption of E6/E6AP complex by GRIM-19. The augment of E6AP ubiquitination by GRIM-19 was detected in vivo and in vitro ubiquitination assay. The effects of GRIM-19-dependent p53 accumulation on cell proliferation, cell cycle, apoptosis were explored by MTT, flow cytometry and transmission electron microscopy respectively. The tumor suppression was detected by xenograft mouse model. CONCLUSION/SIGNIFICANCE: The levels of GRIM-19 and p53 were concurrently down regulated in cervical cancers. The restoration of GRIM-19 can induce ubiquitination and degradation of E6AP, and disrupt the E6/E6AP complex through the interaction of N-terminus of GRIM-19 with both E6 and E6AP, which protected p53 from degradation and promoted cell apoptosis. Tumor xenograft studies also revealed the suppression of p53 degradation in presence of GRIM-19. These data suggest that GRIM-19 can block E6/E6AP complex; and synergistically suppress cervical tumor growth with p53.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Proteínas de Ligação a DNA/metabolismo , NADH NADPH Oxirredutases/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia , Animais , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Feminino , Fase G1 , Humanos , Camundongos , Ligação Proteica , Estabilidade Proteica , Fase de Repouso do Ciclo Celular , Ubiquitinação , Neoplasias do Colo do Útero/ultraestruturaRESUMO
We have previously isolated GRIM-19, a novel growth suppressor, using a genetic method. GRIM-19 ablates cell growth by inhibiting the transcription factor signal transducer and activator of transcription 3 (STAT3). Up-regulation of STAT3 and growth promotion were observed in a number of human tumors. Although the tumor-suppressive actions of GRIM-19 are known, the structural elements required for its antitumor actions are not understood. Mutational and protein sequence analyses identified a motif in the N terminus of GRIM-19 that exhibited similarity to certain RNA viral proteins. We show that disruption of specific amino acids within this motif cripples the antitumor actions of GRIM-19. These mutants fail to interact with STAT3 efficiently and consequently do not inhibit growth-promoting gene expression. More importantly, we show that a clinically observed mutation in the N terminus of GRIM-19 also weakened its interaction with STAT3 and antitumor action. Together, these studies identify a major role for the N terminus of GRIM-19 in mediating its tumor-suppressive actions.